CREB Phosphorylation Research Articles

A Non-Invasive and DNA-free Approach to Upregulate Mammalian Voltage-Gated Calcium Channels and Neuronal Calcium Signaling via Terahertz Stimulation.

Mammalian voltage-gated calcium channels (CaV) play critical roles in cardiac excitability, synaptic transmission, and gene transcription. Dysfunctions in CaV are implicated in a variety of cardiac and neurodevelopmental disorders. Current pharmacological approaches to enhance CaV activity are limited by off-target effects, drug metabolism issues, cytotoxicity, and imprecise modulation. Additionally, genetically-encoded channel activators and optogenetic tools are restricted by gene delivery challenges and biosafety concerns. Here a novel terahertz (THz) wave-based method to upregulate CaV1.2, a key subtype of CaV, and boost CaV1-mediated Ca2+ signaling in neurons without introducing exogenous DNA is presented. Using molecular dynamics simulations, it is shown that 42.5 THz (7.05µm, 1418cm-1) waves enhance Ca2+ conductance in CaV1.2 by resonating with the stretching mode of the -COO- group in the selectivity filter. Electrophysiological recordings and Ca2+ imaging confirm that these waves rapidly, reversibly, and non-thermally increase calcium influx of CaV1.2 in HEK293 cells and induce acute Ca2+ signals in neurons. Furthermore, this irradiation upregulates critical CaV1 signals, including CREB phosphorylation and c-Fos expression, in vitro and in vivo, without raising significant biosafety risks. This DNA-free, non-invasive approach offers a promising approach for modulating CaV gating and Ca2+ signaling and treating diseases characterized by deficits in CaV functions.

BCAT1 contributes to the development of TKI-resistant CML.

Although most of chronic myeloid leukemia (CML) patients can be effectively treated by the tyrosine kinase inhibitors (TKIs), such as Imatinib, TKI-resistance still occurs in approximately 15-17% of cases. Although many studies indicate that branched chain amino acid (BCAA) metabolism may contribute to the TKI resistance in CML, the detailed mechanisms remains largely unknown. The cell proliferation, colony formation and in vivo transplantation were used to determined the functions of BCAT1 in leukemogenesis. Quantitative real-time PCR (RT-PCR), western blotting, RNA sequencing, BCAA stimulation in vitro were applied to characterize the underlying molecular mechanism that control the leukemogenic activity of BCAT1-knockdown cells. In this report, we revealed that branched chain amino acid transaminase 1 (BCAT1) is highly enriched in both mouse and human TKI-resistant CML cells. Leukemia was almost completely abrogated upon BCAT1 knockdown during transplantation in a BCR-ABLT315I-induced murine TKI-resistant CML model. Moreover, knockdown of BCAT1 led to a dramatic decrease in the proliferation of TKI-resistant human leukemia cell lines. BCAA/BCAT1 signaling enhanced the phosphorylation of CREB, which is required for maintenance of TKI-resistant CML cells. Importantly, blockade of BCAA/BCAT1 signaling efficiently inhibited leukemogenesis both in vivo and in vitro. These findings demonstrate the role of BCAA/BCAT1 signaling in cancer development and suggest that targeting BCAA/BCAT1 signaling is a potential strategy for interfering with TKI-resistant CML.

Effects of PPARG on the proliferation, apoptosis, and estrogen secretion in goat granulosa cells

As a member of peroxisome proliferator-activated receptor (PPAR) family, PPARG has been reported to be involved in glucolipid metabolism in various species. However, the function of PPARG in estradiol (E2) synthesis, apoptosis, and proliferation in goat ovarian granulosa cells (GCs) is unclear. In this study, we found that goat PPARG was expressed in GCs of all grades of follicles, and localized in the cytoplasm and nucleus of GCs. Transfection of small interfering RNA-PPARG2 (si-PPARG2) decreased E2 synthesis and the abundances of HSD3B and CYP19A1 mRNA and protein. It also promoted cell apoptosis with significant increases in the ratio of BAX/BCL-2 and Caspase3 mRNA and protein. Meanwhile, cell cycle was inhibited by si-PPARG2 transfection, accompanied by decreased mRNA levels of CDK4, CKD6, MYC, CCND1, CCND2, PCNA, and CCNB, increased mRNA level of P53, and decreased protein levels of CDK4, MYC, and CCND1. Furthermore, PPARG interference affected the mRNA and protein abundances of CREB as well as the phosphorylation of CREB but not PKA. In conclusion, our data suggest that PPARG plays an important role in regulating E2 synthesis, cell apoptosis, and proliferation of goat GCs, including the CREB expression and phosphorylation. These results provide evidences for the in-depth study of PPARG in the regulation of goat GCs function.

Tirzepatide, GIP(1-42) and GIP(1-30) display unique signaling profiles at two common GIP receptor variants, E354 and Q354.

Type 2 diabetes (T2D) and obesity are prevalent metabolic disorders affecting millions of individuals worldwide. A new effective therapeutic drug called tirzepatide for the treatment of obesity and T2D is a dual agonist of the GIP receptor and GLP-1 receptor. Tirzepatide is clinically more effective than GLP-1 receptor agonists but the reasons why are not well understood. Tirzepatide reportedly stimulates the GIP receptor more potently than the GLP-1 receptor. However, tirzepatide signaling has not been thoroughly investigated at the E354 (wildtype) or Q354 (E354Q) GIP receptor variants. The E354Q variant is associated increased risk of T2D and lower body mass index. To better understand GIP receptor signaling we characterized the activity of endogenous agonists and tirzepatide at both GIP receptor variants. Using Cos7 cells we examined wildtype and E354Q GIP receptor signaling, analyzing cAMP and IP1 accumulation as well as AKT, ERK1/2 and CREB phosphorylation. GIP(1-42) and GIP(1-30)NH2 displayed equipotent effects on these pathways excluding CREB phosphorylation where GIP(1-30)NH2 was more potent than GIP(1-42) at the E354Q GIP receptor. Tirzepatide favored cAMP signaling at both variants. These findings indicate that tirzepatide is a biased agonist towards Gαs signaling and suggests it equally activates the wildtype and E354Q GIP receptor variants. We also observed differences between the pharmacology of the GIP receptor variants with endogenous peptides, which may help to explain differences in phenotype. These findings contribute to a comprehensive understanding of GIP receptor signaling, and will aid development of therapies combating T2D and obesity.

The β2-adrenergic biased agonist nebivolol inhibits the development of Th17 and the response of memory Th17 cells in an NF-κB-dependent manner.

Adrenergic receptors regulate metabolic, cardiovascular, and immunological functions in response to the sympathetic nervous system. The effect of β2-adrenergic receptor (AR) as a high expression receptor on different subpopulations of T cells is complex and varies depending on the type of ligand and context. While traditional β2-AR agonists generally suppress T cells, they potentially enhance IL-17A production by Th17 cells. The effects of pharmacological drugs that count as biased agonists of AR like nebivolol are not completely understood. We investigated the impact of nebivolol on human memory CD4+ T (Th1, Th2, Th17) cells and polarized naive Th17 cells, highlighting its potential for IL-17A suppression via a non-canonical β2-AR cell signaling pathway. The effects of nebivolol were tested on healthy human peripheral blood mononuclear cells, purified memory Th cells, and polarized naive Th17 cells activated with anti-CD3/anti-CD28/anti-CD2 ImmunoCult reagent. IFN-γ, IL-4, and IL-17A, which are primarily derived from Th1, Th2, and Th17 cells, respectively, were quantified by ELISA and flow cytometry. IL-10 was measured by ELISA. Gene expression of RORC, ADRB1, ADRB2, and ADRB3 was evaluated by qPCR. The ADRB2 gene was knocked out in memory Th cells using CRISPR/Cas9. Protein expression of phosphorylated serine133-CREB and phosphorylated NF-κB p65 was assessed by Western blot. Proliferation was assessed by fluorescent dye loading and flow cytometry. Nebivolol treatment decreased IL-17A and IFN-γ secretion by activated memory Th cells and elevated IL-4 levels. Nebivolol reduced the proportion of IL-17A+ Th cells and downregulated RORC expression. Unlike the β2-AR agonist terbutaline, nebivolol inhibited the shift of naive CD4+ T cells toward the Th17 phenotype. IL-10 and the proliferation index remained unchanged. Nebivolol-treated β2-knockout memory Th cells showed significant inhibition of β2-AR-mediated signaling, evidenced by the absence of IL-17A suppression compared to controls. Phosphorylation of the NF-κB p65 subunit was inhibited by nebivolol, but CREB phosphorylation was not changed, suggesting a selective transcriptional control. The findings demonstrate that nebivolol acts through a β2-AR-mediated signaling pathway, as a distinctive anti-inflammatory agent capable of selectively shifting Th17 cells and suppressing the phosphorylation of NF-κB. This highlights nebivolol's potential for therapeutic interventions in chronic autoimmune conditions with elevated IL-17A levels.

12248 Leptin Signaling Deficiency At The Central Nervous System Reduces Hepatic Glucagon Sensibility Disturbing Liver Amino Acid Metabolism Leading To Hyperaminoacidemia And Hyperglucagonemia In Male Adult Rats

Abstract Disclosure: C. Pintado Losa: None. L. Mazuecos Fernández-Pacheco: None. Ó. Gómez Torres: None. S. Artigas Jerónimo: None. E. Burgos Ramos: None. I. Ramos: None. J. Poveda Colado: None. C. Arribas Mocoroa: None. A. Andres: None. N. Gallardo: None. Leptin regulates glucagon secretion, but the contribution of central leptin resistance to circulating glucagon levels and hepatic glucagon signaling is poorly studied. To analyze the effects of deficient hypothalamic leptin signaling on glucagon levels and hepatic glucagon signaling, we infused intracerebroventricularly a rat-specific leptin receptor antagonist SLA (0,2 µg/day) for 21 days to male 3-month-old Wistar rats (n=12) or PBS (n=12). At day 21 of treatment and 30 min before sacrifice, a glucagon challenge (100 µg/kg) was performed in 16h fasted rats. After sacrifice, blood and liver samples were collected for analysis. Serum hormones, urea, and total and branched-chain amino acids (BCAA) levels were determined using commercial kits and by RP-HPLC. Hepatic neutral lipid was extracted and quantified using an enzymatic kit and the hepatic content of mRNA and proteins involved in lipid and amino acid metabolism were measured by RT-PCR and Western-Blot. Additionally, the HOMA-IR and glucagon-alanine index were determined in all rats. Blocking central leptin signaling led to impaired fat oxidation and triglyceride export capacity in the liver, resulting in a 50% increase in hepatic triglyceride content in SLA-treated rats. Glucagonemia increased in SLA-treated rats, indicating impaired hepatic glucagon sensitivity. In fact, in response to exogenous glucagon, the hepatic phosphorylation of CREB was reduced by 55% in SLA-infused compared to control rats. Importantly, SLA treatment significantly decreases the expression and the activity of BCKDH (branched-chain alpha-keto acid dehydrogenase) in the liver, leading to a reduction in the BCAA degradation in SLA-treated rats, hyperaminoacidemia, and decreased serum urea levels. In conclusion, deficient hypothalamic leptin signaling alters glucose, lipid, and amino acid homeostasis, highlighting the relevance of central leptin action in the control of peripheral metabolism and glucagon sensitivity. Funding: Work supported by Ministerio de Ciencia, Innovación y Universidades under project grants RTI2018-098643-B-I00, PID2021-128243OB-I00, and from the University of Castilla-La Mancha and the European Regional Development Fund (FEDER) under project grant 2023- GRIN-34280. Presentation: 6/1/2024

Mammalian D-Cysteine controls insulin secretion in the pancreas

D-amino acids are being recognized as important molecules in mammals with function. This is a first identification of endogenous D-cysteine in mammalian pancreas. D-cysteine is synthesized by serine racemase (SR) and SR−/− mice produce 6–10 fold higher levels of insulin in the pancreas and plasma including higher glycogen and ketone bodies in the liver. The excess insulin is stored as amyloid in secretory vesicles and exosomes. In glucose stimulated insulin secretion in mouse and human islets, equimolar amount of D-cysteine showed higher inhibition of insulin secretion compared to D-serine, another closely related stereoisomer synthesized by SR. In mouse models of diabetes (Streptozotocin (STZ) and Non Obese Diabetes (NOD) and human pancreas, the diabetic state showed increased expression of D-cysteine compared to D-serine followed by increased expression of SR. SR−/− mice show decreased cAMP in the pancreas, lower DNA methyltransferase enzymatic and promoter activities followed by reduced phosphorylation of CREB (S133), resulting in decreased methylation of the Ins1 promoter. D-cysteine is efficiently metabolized by D-amino acid oxidase and transported by ASCT2 and Asc1. Dietary supplementation with methyl donors restored the high insulin levels and low DNMT enzymatic activity in SR−/− mice. Our data show that endogenous D-cysteine in the mammalian pancreas is a regulator of insulin secretion.

Role of canonical and non-canonical cAMP sources in CRHR2α-dependent signaling.

Hippocampal neurons exhibit activation of both the conventional transmembrane adenylyl cyclases (tmACs) and the non-canonical soluble adenylyl cyclase (sAC) as sources of cyclic AMP (cAMP). These two cAMP sources play crucial roles in mediating signaling pathways downstream of CRHR1 in neuronal and neuroendocrine contexts. In this study, we investigate the involvement of both cAMP sources in the molecular mechanisms triggered by CRHR2α. Here we provide evidence demonstrating that UCN1 and UCN3 exert a neuritogenic effect on HT22-CRHR2α cells, which is solely dependent on the cAMP pool generated by sAC and PKA activity but independent of ERK1/2 activation. Through the characterization of the effectors implicated in neurite elongation, we found that CREB phosphorylation and c-Fos induction rely on PKA activity and ERK1/2 phosphorylation, underscoring the critical role of signaling pathway regulation. These findings strengthen the concept that localized cAMP microdomains actively participate in the regulation of these signaling processes.

Pde3a and Pde3b regulation of murine pulmonary artery smooth muscle cell growth and metabolism.

A role for metabolically active adipose tissue in pulmonary hypertension (PH) pathogenesis is emerging. Alterations in cellular metabolism in metabolic syndrome are triggers of PH-related vascular dysfunction. Metabolic reprogramming in proliferative pulmonary vascular cells causes a metabolic switch from oxidative phosphorylation to glycolysis. PDE3A and PDE3B subtypes in the regulation of metabolism in pulmonary artery smooth muscle cells (PASMC) are poorly understood. We previously found that PDE3A modulates the cellular energy sensor, AMPK, in human PASMC. We demonstrate that global Pde3a knockout mice have right ventricular (RV) hypertrophy, elevated RV systolic pressures, and metabolic dysfunction with elevated serum free fatty acids (FFA). Therefore, we sought to delineate Pde3a/Pde3b regulation of metabolic pathways in PASMC. We found that PASMC Pde3a deficiency, and to a lesser extent Pde3b deficiency, downregulates AMPK, CREB and PPARγ, and upregulates pyruvate kinase dehydrogenase expression, suggesting decreased oxidative phosphorylation. Interestingly, siRNA Pde3a knockdown in adipocytes led to elevated FFA secretion. Furthermore, PASMC exposed to siPDE3A-transfected adipocyte media led to decreased α-SMA, AMPK and CREB phosphorylation, and greater viable cell numbers compared to controls under the same conditions. These data demonstrate that deficiencies of Pde3a and Pde3b alter pathways that affect cell growth and metabolism in PASMC.

Neuroprotection by 4R-cembranoid against Gulf War Illness-related chemicals is mediated by ERK, PI3K, and CaMKII pathways

Gulf War Illness (GWI) has been consistently linked to exposure to pyridostigmine (PB), N,N-Diethyl-meta-toluamide (DEET), permethrin (PER), and traces of sarin. In this study, diisopropylfluorophosphate (DFP, sarin surrogate) and the GWI-related chemicals were found to reduce the number of functionally active neurons in rat hippocampal slices. These findings confirm a link between GWI neurotoxicants and N-Methyl-D-Aspartate (NMDA)-mediated excitotoxicity, which was successfully reversed by Edelfosine (a phospholipase Cβ (PLCβ3) inhibitor) and Flupirtine (a KCNQ/M (Kv7) channel agonist).To test whether 4R-cembranoid (4R), a nicotinic α7 acetylcholinesterase receptor (α7AChR) modulator known for its neuroprotective properties, can restore hippocampal neurons from glutamate-induced neurotoxicity, we exposed rat hippocampal slices with DFP for 10 min followed by 60 min treatment with 4R. We investigated the 4R mechanisms of neuroprotection after preincubation with LY294002, PD98059, and KN-62. The inhibition of the phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinase kinase (MEK1/2), and calcium/calmodulin-dependent protein kinase (CaMKII) abrogated the protective effect of 4R against DFP-induced neurotoxicity. In separate experiments, after incubation with DFP, followed by 4R for 1 hr., cellular extracts were prepared for Western blotting of phospho-Akt, phospho-GSK3β, phosphorylated extracellular signal-regulated kinase (ERK)1/2, CaMKII and cAMP response element-binding protein (CREB). Our results show that DFP induces neuronal dysfunction by dephosphorylation, while 4R restores the phosphorylation of Akt, GSK3, ERK1/2, CREB, and CaMKII. Moreover, our proteomics analysis supported the notion that 4R activates additional signaling pathways related to enhancing neuronal signaling, synaptic plasticity, and apoptotic inhibition to promote cell survival against DFP, offering biomarkers for developing treatment against GWI.

ZBED3 exacerbates hyperglycemia by promoting hepatic gluconeogenesis through CREB signaling

BackgroundElevated hepatic glucose production (HGP) is a prominent manifestation of impaired hepatic glucose metabolism in individuals with diabetes. Increased hepatic gluconeogenesis plays a pivotal role in the dysregulation of hepatic glucose metabolism and contributes significantly to fasting hyperglycemia in diabetes. Previous studies have identified zinc-finger BED domain-containing 3 (ZBED3) as a risk gene for type 2 diabetes (T2DM), and its single nucleotide polymorphism (SNPs) is closely associated with the fasting blood glucose level, suggesting a potential correlation between ZBED3 and the onset of diabetes. This study primarily explores the effect of ZBED3 on hepatic gluconeogenesis and analyzes the relevant signaling pathways that regulate hepatic gluconeogenesis. MethodsThe expression level of ZBED3 was assessed in the liver of insulin-resistant (IR)-related disease. RNA-seq and bioinformatics analyses were employed to examine the ZBED3-related pathway that modulated HGP. To investigate the role of ZBED3 in hepatic gluconeogenesis, the expression of ZBED3 was manipulated by upregulation or silencing using adeno-associated virus (AAV) in mouse primary hepatocytes (MPHs) and HHL-5 cells. In vivo, hepatocyte-specific ZBED3 knockout mice were generated. Moreover, AAV8 was employed to achieve hepatocyte-specific overexpression and knockdown of ZBED3 in C57BL/6 and db/db mice. Immunoprecipitation and mass spectrometry (IP-MS) analyses were employed to identify proteins that interacted with ZBED3. Co-immunoprecipitation (co-IP), glutathione S-transferase (GST) - pulldown, and dual-luciferase reporter assays were conducted to further elucidate the underlying mechanism of ZBED3 in regulating hepatic gluconeogenesis. ResultsThe expression of ZBED3 in the liver of IR-related disease models was found to be increased. Under the stimulation of glucagon, ZBED3 promoted the expression of hepatic gluconeogenesis-related genes PGC1A, PCK1, G6PC, thereby increasing HGP. Consistently, the rate of hepatic gluconeogenesis was found to be elevated in mice with hepatocyte-specific overexpression of ZBED3 and decreased in those with ZBED3 knockout. Additionally, the knockdown of ZBED3 in the liver of db/db mice resulted in a reduction in hepatic gluconeogenesis. Moreover, the study revealed that ZBED3 facilitated the nuclear translocation of protein arginine methyltransferases 5 (PRMT5) to influence the regulation of PRMT5-mediated symmetrical dimethylation of arginine (s-DMA) of cyclic adenosine monophosphate (cAMP) response element binding protein (CREB), which in turn affects the phosphorylation of CREB and ultimately promotes HGP. ConclusionsThis study indicates that ZBED3 promotes hepatic gluconeogenesis and serves as a critical regulator of the progression of diabetes.

CDNF Exerts Anxiolytic, Antidepressant-like, and Procognitive Effects and Modulates Serotonin Turnover and Neuroplasticity-Related Genes.

Cerebral dopamine neurotrophic factor (CDNF) is an unconventional neurotrophic factor because it does not bind to a known specific receptor on the plasma membrane and functions primarily as an unfolded protein response (UPR) regulator in the endoplasmic reticulum. Data on the effects of CDNF on nonmotor behavior and monoamine metabolism are limited. Here, we performed the intracerebroventricular injection of a recombinant CDNF protein at doses of 3, 10, and 30 μg in C57BL/6 mice. No adverse effects of the CDNF injection on feed and water consumption or locomotor activity were observed for 3 days afterwards. Decreases in body weight and sleep duration were transient. CDNF-treated animals demonstrated improved performance on the operant learning task and a substantial decrease in anxiety and behavioral despair. CDNF in all the doses enhanced serotonin (5-HT) turnover in the murine frontal cortex, hippocampus, and midbrain. This alteration was accompanied by changes in the mRNA levels of the 5-HT1A and 5-HT7 receptors and in monoamine oxidase A mRNA and protein levels. We found that CDNF dramatically increased c-Fos mRNA levels in all investigated brain areas but elevated the phosphorylated-c-Fos level only in the midbrain. Similarly, enhanced CREB phosphorylation was found in the midbrain in experimental animals. Additionally, the upregulation of a spliced transcript of XBP1 (UPR regulator) was detected in the midbrain and frontal cortex. Thus, we can hypothesize that exogenous CDNF modulates the UPR pathway and overall neuronal activation and enhances 5-HT turnover, thereby affecting learning and emotion-related behavior.

MicroRNA-195-5p Inhibits Intracerebral Hemorrhage-Induced Inflammatory Response and Neuron Cell Apoptosis.

Intracerebral hemorrhage (ICH) is a severe condition characterized by bleeding within brain tissue. Primary brain injury in ICH results from a mechanical insult caused by blood accumulation, whereas secondary injury involves inflammation, oxidative stress, and disruption of brain physiology. miR-195-5p may participate in ICH pathology by regulating cell proliferation, oxidative stress, and inflammation. Therefore, we assessed the performance of miR-195-5p in alleviating ICH-induced secondary brain injury. ICH was established in male Sprague-Dawley rats (7 weeks old, 200-250 g) via the stereotaxic intrastriatal injection of type IV bacterial collagenase, after which miR-195-5p was administered intravenously. Neurological function was assessed using corner turn and forelimb grip strength tests. Protein expression was assessed by western blotting and ELISA. The miR-195-5p treatment significantly improved neurological function; modulated macrophage polarization by promoting anti-inflammatory marker (CD206 and Arg1) production and inhibiting pro-inflammatory marker (CD68 and iNOS) production; enhanced Akt signalling, reduced oxidative stress by increasing Sirt1 and Nrf2 levels, and attenuated inflammation by decreasing NF-κB activation; inhibited apoptosis via increased Bcl-2 and decreased cleaved caspase-3 levels; and regulated synaptic plasticity by modulating NMDAR2A, NMDAR2B, BDNF, and TrkB expression and ERK and CREB phosphorylation. In conclusion, miR-195-5p exerts neuroprotective effects in ICH by reducing inflammation and oxidative stress, inhibiting apoptosis, and restoring synaptic plasticity, ultimately restoring behavioral recovery, and represents a promising therapeutic agent that warrants clinical studies.

Upregulation of Phosphodiesterase 7A Contributes to Concurrent Pain and Depression via Inhibition of cAMP-PKA-CREB-BDNF Signaling and Neuroinflammation in the Hippocampus of Mice.

Phosphodiesterases (PDEs) are enzymes that catalyze the hydrolysis of cyclic adenosine monophosphate AMP (cAMP) and/or cyclic guanosine monophosphate (cGMP). PDE inhibitors can mitigate chronic pain and depression when these disorders occur individually; however, there is limited understanding of their role in concurrent chronic pain and depression. We aimed to evaluate the mechanisms of action of PDE using 2 mouse models of concurrent chronic pain and depression. C57BL/6J mice were subjected to partial sciatic nerve ligation (PSNL) to induce chronic neuropathic pain or injected with complete Freund's adjuvant (CFA) to induce inflammatory pain, and both animals showed depression-like behavior. First, we determined the change in PDE expression in both animal models. Next, we determined the effect of PDE7 inhibitor BRL50481 or hippocampal PDE7A knockdown on PSNL- or CFA-induced chronic pain and depression-like behavior. We also investigated the role of cAMP-protein kinase A (PKA)-cAMP response element binding protein (CREB)-brain-derived neurotrophic factor (BDNF) signaling and neuroinflammation in the effect of PDE7A inhibition on PSNL- or CFA-induced chronic pain and depression-like behavior. This induction of chronic pain and depression in the 2 animal models upregulated hippocampal PDE7A. Oral administration of PDE7 inhibitor, BRL50481, or hippocampal PDE7A knockdown significantly reduced mechanical hypersensitivity and depression-like behavior. Hippocampal PDE7 inhibition reversed PSNL- or CFA-induced downregulation of cAMP and BDNF and the phosphorylation of PKA, CREB, and p65. cAMP agonist forskolin reversed these changes and caused milder behavioral symptoms of pain and depression. BRL50481 reversed neuroinflammation in the hippocampus in PSNL mice. Hippocampal PDE7A mediated concurrent chronic pain and depression in both mouse models by inhibiting cAMP-PKA-CREB-BDNF signaling. Inhibiting PDE7A or activating cAMP-PKA-CREB-BDNF signaling are potential strategies to treat concurrent chronic pain and depression.

Lipoxin A4 modulates the PKA/CREB and NF-κB signaling pathway to mitigate chondrocyte catabolism and apoptosis in temporomandibular joint osteoarthritis

Temporomandibular joint osteoarthritis (TMJ-OA) is characterized by the degradation of the extracellular matrix (ECM) in cartilage and the apoptosis of chondrocytes, which is caused by inflammation and disruptions of chondrocyte metabolism and inflammation. Lipoxin A4 (LXA4), a specialized pro-resolving mediator, has been shown to inhibit inflammation and regulate the balance between ECM synthesis and degradation. However, the therapeutic effects of LXA4 on TMJ-OA and its underlying mechanisms remain unclear. Interleukin-1 beta (IL-1β)-induced chondrocyte and surgically induced TMJ-OA rat models were established in this study. The viability of chondrocytes treated with LXA4 was evaluated with the cell counting kit-8 (CCK-8) assay, while protein levels were assessed by western blot analysis, and the apoptosis rate was evaluated with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labelling (TUNEL) staining. Histological analysis was conducted to evaluate the impact of LXA4 on cartilage degradation in TMJ-OA rat models. In vitro, the qRT-PCR and western blot analysis demonstrated that LXA4 facilitated the upregulation of collagen proteins (Collagen II) and decreased expression of matrix metalloproteinases (MMP-3, and MMP-13) associated with ECM modulation. LXA4 enhanced the TMJ-OA chondrocyte viability and decreased apoptotic rate. In vivo, histology and immunohistochemistry (IHC) analysis revealed that intraperitoneal injection of LXA4 contributed to the amelioration of chondrocyte injuries and deceleration of TMJ-OA. Transcriptomic sequencing revealed that cAMP signaling pathway was up-regulated and NF-κB signaling pathway was down-regulated in LXA4 treated group. LXA4 inhibited the phosphorylation of P65 and inhibitor of nuclear factor kappa B (IκBα) proteins while enhancing the phosphorylation PKA and CREB. This study demonstrates the potential of LXA4 as a therapeutic agent for suppressing chondrocyte catabolism and apoptosis by increasing PKA/CREB activity and decreasing NF-κB signaling.

New BDNF and NT-3 Cyclic Mimetics Concur with Copper to Activate Trophic Signaling Pathways as Potential Molecular Entities to Protect Old Brains from Neurodegeneration.

A low level of Neurotrophins (NTs), their Tyrosine Kinase Receptors (Trks), Vascular Endothelial Growth Factors (VEGFs) and their receptors, mainly VEGFR1 and VEGFR2, characterizes AD brains. The use of NTs and VEGFs as drugs presents different issues due to their low permeability of the blood-brain barrier, the poor pharmacokinetic profile, and the relevant side effects. To overcome these issues, different functional and structural NT mimics have been employed. Being aware that the N-terminus domain as the key domain of NTs for the binding selectivity and activation of Trks and the need to avoid or delay proteolysis, we herein report on the mimicking ability of two cyclic peptide encompassing the N-terminus of Brain Derived Growth Factor (BDNF), (c-[HSDPARRGELSV-]), cBDNF(1-12) and of Neurotrophin3 (NT3), (c-[YAEHKSHRGEYSV-]), cNT3(1-13). The two cyclic peptide features were characterized by a combined thermodynamic and spectroscopic approach (potentiometry, NMR, UV-vis and CD) that was extended to their copper(II) ion complexes. SH-SY5Y cell assays show that the Cu2+ present at the sub-micromolar level in the complete culture media affects the treatments with the two peptides. cBDNF(1-12) and cNT3(1-13) act as ionophores, induce neuronal differentiation and promote Trks and CREB phosphorylation in a copper dependent manner. Consistently, both peptide and Cu2+ stimulate BDNF and VEGF expression as well as VEGF release; cBDNF(1-12) and cNT3(1-13) induce the expression of Trks and VEGFRs.

Areca nut-induced metabolic reprogramming and M2 differentiation promote OPMD malignant transformation

BackgroundBetel quid and its major ingredient, areca nut, are recognized by IARC as major risk factors in oral cancer development. Areca nut extract (ANE) exposure has been linked to OPMD progression and malignant transformation to OSCC. However, the detailed mechanism through which ANE acts on other cell types in the oral microenvironment to promote oral carcinogenesis remains elusive.MethodsImmunoprofiling of macrophages associated with OPMD and OSCC was carried out by immunohistochemical and immunofluorescence staining. Phosphokinase and cytokine arrays and western blotting were performed to determine the underlying mechanisms. Transwell assays were used to evaluate the migration-promoting effect of ANE. Hamster model was finally applied to confirm the in vivo effect of ANE.ResultsWe reported that M2 macrophages positively correlated with oral cancer progression. ANE induced M2 macrophage differentiation, CREB phosphorylation and VCAM-1 secretion and increased mitochondrial metabolism. Conditioned medium and VCAM-1 from ANE-treated macrophages promoted migration and mesenchymal phenotypes in oral precancer cells. In vivo studies showed that ANE enhanced M2 polarization and related signaling pathways in the oral buccal tissues of hamsters.ConclusionOur study provides novel mechanisms for areca nut-induced oral carcinogenesis, demonstrating that areca nut promotes M2 macrophage differentiation and secretion of oncogenic cytokines that critically activate malignant transformation of oral premalignant cells.

Therapeutic Options of Crystallin Mu and Protein Disulfide Isomerase A3 for Cuprizone-Induced Demyelination in Mouse Hippocampus

This study investigates the changes in hippocampal proteomic profiles during demyelination and remyelination using the cuprizone model. Employing two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry for protein profiling, we observed significant alterations in the expression of ketimine reductase mu-crystallin (CRYM) and protein disulfide isomerase A3 precursor (PDIA3) following exposure to and subsequent withdrawal from cuprizone. Immunohistochemical staining validated these protein expression patterns in the hippocampus, revealing that both PDIA3 and CRYM were downregulated in the hippocampal CA1 region during demyelination and upregulated during remyelination. Additionally, we explored the potential protective effects of CRYM and PDIA3 against cuprizone-induced demyelination by synthesizing cell-permeable Tat peptide-fusion proteins (Tat-CRYM and Tat-PDIA3) to facilitate their crossing through the blood–brain barrier. Our results indicated that administering Tat-CRYM and Tat-PDIA3 mitigated the reduction in proliferating cell and differentiated neuroblast counts compared to the group receiving cuprizone alone. Notably, Tat-PDIA3 demonstrated significant effects in enhancing myelin basic protein expression alongside phosphorylation of CREB in the hippocampus, suggesting its potential therapeutic role in the prevention or treatment of demyelination, and by extension, in conditions such as multiple sclerosis.

Exploring novel A2AAR antagonists: Design, synthesis, and evaluation of 2,6,9-trisubstituted purine derivatives as promising antifibrotic agents

A series of 2,6,9-trisubstituted purine derivatives were designed and synthesized with diverse chemical moieties. Through a comprehensive biological evaluation, we identified 4-(6-(methylamino)-2-(phenylethynyl)-9H-purin-9-yl)phenol (6a) as a promising A2AAR antagonist with potent antifibrotic properties. Compound 6a demonstrated significant efficacy in inhibiting CRE promoter activity and in reducing the expression of fibrogenic marker proteins and downstream effectors of A2AAR activation, surpassing the A2AAR antagonist ZM241385 and initial screening hits, 9-benzyl-N-methyl-2-(phenylethynyl)-9H-purin-6-amine (5a) and 9-((benzyloxy)methyl)-N-methyl-2-(phenylethynyl)-9H-purin-6-amine (5j). Further validation revealed that compound 6a effectively inhibited fibrogenic marker proteins induced by A2AAR overexpression or TGF-β1 treatment in hepatic stellate cells, alongside reducing PKA and CREB phosphorylation. These findings suggest that compound 6a exerts its antifibrotic action by modulating the cAMP/PKA/CREB pathway through A2AAR inhibition. Overall, our study provides valuable insights for the development of novel therapeutics that target hepatic fibrosis through A2AAR antagonism.

CysLT receptor-mediated NOX2 activation is required for IL-8 production in HMC-1 cells induced by Trichomonas vaginalis-derived secretory products.

Trichomoniasis is caused by a sexually transmitted flagellate protozoan parasite Trichomonas vaginalis. T. vaginalis-derived secretory products (TvSP) contain lipid mediators such as leukotriene B4 (LTB4) and various cysteinyl leukotrienes (CysLTs) which included LTC4, LTD4, and LTE4. However, the signaling mechanisms by which T. vaginalis-induced CysLTs stimulate interleukin (IL)-8 production in human mast cells remain unclear. In this study, we investigated these mechanisms in human mast cells (HMC-1). Stimulation with TvSP resulted in increased intracellular reactive oxygen species (ROS) generation and NADPH oxidase 2 (NOX2) activation compared to unstimulated cells. Pre-treatment with NOX2 inhibitors such as diphenyleneiodonium chloride (DPI) or apocynin significantly reduced ROS production in TvSP-stimulated HMC-1 cells. Additionally, TvSP stimulation increased NOX2 protein expression and the translocation of p47phox from the cytosol to the membrane. Pretreatment of HMC-1 cells with PI3K or PKC inhibitors reduced TvSP-induced p47phox translocation and ROS generation. Furthermore, NOX2 inhibitors or NOX2 siRNA prevented CREB phosphorylation and IL-8 gene expression or protein secretion induced by TvSP. Pretreatment with a CysLTR antagonist significantly inhibited TvSP-induced ROS production, CREB phosphorylation, and IL-8 production. These results indicate that CysLT-mediated activation of NOX2 plays a crucial role in ROS-dependent IL-8 production in human mast cells stimulated by T. vaginalis-secreted CysLTs. These findings enhance our understanding of the inflammatory response in trichomoniasis and may inform the development of targeted therapies to mitigate this response.